A SnapShot of Ubiquitin Chain Elongation

Kovacev, Jordan; Wu, Ken; Spratt, Donald E.; Chong, Robert; Lee, Chan; Nayak, Jaladhi; Shaw, Gary S.; Pan, Zhen‐Qiang

doi:10.1074/jbc.m113.530576

Cited by 11 publications

(9 citation statements)

References 40 publications

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“…For example, in the case of K48-linked polyubiquitin, a length of four is generally considered optimal for molecular recognition of the 26 S proteasome ( 12 ), while the nuclear protein localization protein 4 is selective for K48-linked chains longer than six ( 13 ). It was reported that K48-linked tetraubiquitin (Ub 4 ) slows down further ubiquitination ( 14 – 16 ), while this is not the case for K63-linked Ub 4 ( 16 ).…”

Section: Introductionmentioning

confidence: 99%

The dynamics of linear polyubiquitin

et al. 2020

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Polyubiquitin chains are flexible multidomain proteins, whose conformational dynamics enable them to regulate multiple biological pathways. Their dynamic is determined by the linkage between ubiquitins and by the number of ubiquitin units. Characterizing polyubiquitin behavior as a function of their length is hampered because of increasing system size and conformational variability. Here, we introduce a new approach to efficiently integrating small-angle x-ray scattering with simulations allowing us to accurately characterize the dynamics of linear di-, tri-, and tetraubiquitin in the free state as well as of diubiquitin in complex with NEMO, a central regulator in the NF-κB pathway. Our results show that the behavior of the diubiquitin subunits is independent of the presence of additional ubiquitin modules and that the dynamics of polyubiquitins with different lengths follow a simple model. Together with experimental data from multiple biophysical techniques, we then rationalize the 2:1 NEMO:polyubiquitin binding.

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Section: Introductionmentioning

confidence: 99%

The dynamics of linear polyubiquitin

et al. 2020

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“…For this purpose, SCF βTrCP /IκBα (EE)–Ub (I647) and Cdc34∼Ub (K48R/I555), preassembled in separate tubes, were each treated with apyrase to deplete ATP prior to mixing for ubiquitination. The effectiveness of ATP depletion by the apyrase protocol was validated previously ( 24 ). The results revealed that FRET plateaued at 30 min, utilizing about 20% of substrate input ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To confirm this effect, we have performed competition experiments with the phospho-β catenin degron peptide as a competitor. Previous crystallographic and biochemical studies have shown binding of the phospho-β catenin degron peptide to βTrCP ( 21 ) and ability of this peptide to support ubiquitination by SCF βTrCP ( 24 ). As shown, the phospho-β catenin degron peptide was able to effectively compete against the fluorescent IκBα substrate by blocking the FRET IκBα diubiquitination ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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A new FRET-based platform to track substrate ubiquitination by fluorescence

Wu¹,

Ching²,

Chong

et al. 2021

Journal of Biological Chemistry

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Post-translational modification of protein by ubiquitin (Ub) alters the stability, subcellular location, or function of the target protein, thereby impacting numerous biological processes and directly contributing to myriad cellular defects or disease states, such as cancer. Tracking substrate ubiquitination by fluorescence provides opportunities for advanced reaction dynamics studies and for translational research including drug discovery. However, fluorescence-based techniques in ubiquitination studies remain underexplored at least partly because of challenges associated with Ub chain complexity and requirement for additional substrate modification. Here we describe a general strategy, FRET diubiquitination, to track substrate ubiquitination by fluorescence. This platform produces a uniform di-Ub product depending on specific interactions between a substrate and its cognate E3 Ub ligase. The diubiquitination creates proximity between the Ub-linked donor and acceptor fluorophores, respectively, enabling energy transfer to yield a distinct fluorescent signal. FRET diubiquitination relies on Ub–substrate fusion, which can be implemented using either one of the two validated strategies. Method 1 is the use of recombinant substrate–Ub fusion, applicable to all substrate peptides that can bind to E3. Method 2 is a chemoenzymatic ligation approach that employs synthetic chemistry to fuse Ub with a substrate peptide containing desired modification. Taken together, our new FRET-based diubiquitination system provides a timely technology of potential to advance both basic research and translation sciences.

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“…Most previous measurements of ubiquitination rates relied on quantification of protein bands from Western blots (e.g. [21][22][23][24]). While reliable, SDS-PAGE and antibody detection steps are time-consuming and expensive.…”

Section: Introductionmentioning

confidence: 99%

A generalin vitroassay to study enzymatic activities of the ubiquitin system

Chong

Zuo

Jiang

et al. 2019

Preprint

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The ubiquitin (Ub) system regulates a wide range of cellular signaling pathways. Several hundred E1, E2 and E3 enzymes are together responsible for the conjugation of Ub to cellular proteins, thereby controlling biological activities.Due to the numerous enzymes and processes involved in ubiquitination, studies on the molecular mechanisms have been challenging. We here report a novel FRET-based assay to study the kinetics of ubiquitination. FRET is established between binding of fluorophore-labeled Ub to eGFP-tagged ZnUBP, a domain that exclusively binds unconjugated Ub. We name this assay the Free Ub Sensor System (FUSS). Using Uba1, UbcH5 and CHIP as model enzymes, we demonstrate that ubiquitination rates can be measured by FUSS using the rate of change in the FRET efficiency over time. Treatment with USP21, a deubiquitinase, removes ubiquitination on CHIP and leads to increased FRET efficiency, confirming the reversibility of the assay. Incubation with tau, a substrate of CHIP, increases the overall ubiquitination activity. We further use this assay to measure the relationship between increasing E1, E2 or E3 concentrations and ubiquitination rates, revealing that Uba1 activity is the ratelimiting step. Our data together demonstrate the versatile applications of a novel ubiquitination assay that does not require labeling of E1, E2, E3 or substrates, and is thus potentially compatible with any E1-E2-E3 combinations. (214 words)

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A SnapShot of Ubiquitin Chain Elongation

Cited by 11 publications

References 40 publications

The dynamics of linear polyubiquitin

The dynamics of linear polyubiquitin

A new FRET-based platform to track substrate ubiquitination by fluorescence

A generalin vitroassay to study enzymatic activities of the ubiquitin system

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